Biology subjects

Vanoosthuyse, V.

Publications and source records attributed to Vanoosthuyse, V..

6 recordsLinked to original sources

bioRxiv2026

The rapid delivery of RNase H variants reveals distinct classes of RNA:DNA hybrids

Multiple lines of evidence suggest that RNase H1-sensitive RNA:DNA hybrids (thereafter referred to as hybrids) could disrupt the stability and restart of stalled replication forks. The prolonged over-expression of RNase H1 is the most popular method to manipulate hybrids in situ but it cannot discriminate between direct and indirect effects resulting from their loss. To overcome this limitation, we developed iCoRD (in Cellulo RNase H Delivery) to rapidly deliver comparable amounts of either Escherichia coli RNase H1 (RnhA) or control proteins in live cells, with a negligible impact on the transcriptome. iCoRD-delivered RnhA recognises hybrids in situ, is enriched in the vicinity of active replication forks and rescues replication fork progression in different stress conditions. While iCoRD-delivered RnhA had a clear impact on stress-induced, fork-proximal hybrids and R-ChIP signals, it had no effect on DRIP or S9.6 Cut&Tag signals, even when these were induced by stress. Our results strongly suggest the existence of distinct populations of hybrids that are best mapped by different methods and are differently sensitive to RNase H1 activity in situ.

molecular biology↗

bioRxiv2025

Rapid delivery of RNase H1 enables replication fork progression under stress independently of R-loop resolution

The over-expression of RNase H1 enables the progression of replication forks under stress and safeguards genome stability. The most common interpretation of these observations is that RNase H1 over-expression removes R-loops, whose formation would hinder replication fork progression. Here we challenge this model using an innovative strategy to rapidly deliver ready-made Escherichia coli RNase H1 (RnhA) in live cells. We show that RnhA is enriched in the vicinity of active replication forks and that it rescues replication fork progression in different stress conditions. However, RnhA had no impact on R-loops mapped by the DRIP or Cut&Tag methods. Our results reveal the existence of distinct populations of RNA:DNA hybrids that are differently sensitive to RnhA and invites new interpretations of well-established observations. HIGHLIGHTSO_LIRapid delivery of R-loop sensors and regulators in live cells C_LIO_LIRapid delivery of RnhA rescues replication fork progression in stress conditions C_LIO_LIRapid delivery of RnhA does not impact R-loops C_LIO_LIDRIP detects RNA:DNA species insensitive to RNase H1 in cells C_LI

molecular biology↗

bioRxiv2020

RNA polymerase backtracking drives the accumulation of fission yeast condensin at active genes

The mechanisms leading to the accumulation of the SMC complexes condensins around specific transcription units remain unclear. Observations made in bacteria suggested that RNA polymerases (RNAP) constitute an obstacle to SMC translocation, particularly when RNAP and SMC travel in opposite directions. Here we show in fission yeast that gene termini harbour intrinsic condensin-accumulating features whatever the orientation of transcription, which we attribute to the frequent backtracking of RNAP at gene ends. Consistent with this, to relocate backtracked RNAP2 from gene termini to gene bodies was sufficient to cancel the accumulation of condensin at gene ends and to redistribute it evenly within transcription units, indicating that RNAP backtracking may play a key role in positioning condensin. Formalization of this hypothesis in a mathematical model suggests that the inclusion of a sub-population of RNAP with longer dwell-times is essential to fully recapitulate the distribution profiles of condensin around active genes. Taken together, our data strengthen the idea that dense arrays of proteins tightly-bound to DNA alter the distribution of condensin on chromosomes.

cell biology↗

bioRxiv2019

The sequence of the extruded non-template strand determines the architecture of R-loops.

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the sequence of the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

biophysics↗

bioRxiv2019

Sen1 is required for RNA Polymerase III transcription termination in an R-loop independent manner.

R-loop disassembly by the human helicase Senataxin contributes to genome stability and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin-mediated R-loop disassembly also contributes to transcription termination at other classes of genes has remained unclear. Here we show in fission yeast that SenataxinSen1 promotes efficient termination of RNA Polymerase III (RNAP3) transcription in vivo. In the absence of SenataxinSen1, RNAP3 accumulates downstream of the primary terminator at RNAP3-transcribed genes and produces long exosome-sensitive 3-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that SenataxinSen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Senataxin is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

molecular biology↗

bioRxiv2017

The affinity of the S9.6 antibody for double-stranded RNAs impacts the mapping of R-loops in fission yeast.

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

molecular biology↗